Construction of a doxycycline inducible adipogenic lentiviral expression system.

To provide a tool for research on regulating adipocyte differentiation, tetracycline inducible (Tet on) lentiviral expression vectors under the control of an adipose-specific promoter were constructed. The lowest basal expression in the absence of doxycycline and most efficient dose-dependent, doxycycline-induced transient overexpression was observed using vectors constructed with a combination of Tetracycline Responsive Element (TRE) and reverse tetracycline-controlled TransActivator advanced (rtTAadv), transfected in white (3T3-L1) and brown (HIB-1B) preadipocytes cell lines. The results demonstrate that doxycycline adipogenic inducible expression can be achieved using a pLenti TRE / rtTA adv under the control of the truncated aP2 promoter in HIB-1B preadipocytes.

The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm.

Compositional analysis of nuclear pore complexes (NPCs) is nearing completion, and efforts are now focused on understanding how these protein machines work. Recent analysis of soluble transport factor interactions with NPC proteins reveals distinct and overlapping pathways for movement between the nucleus and cytoplasm. New fluorescence- and microscopy-based strategies have been used to monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during transport.

Development and modeling of arsenic-trioxide-loaded thermosensitive liposomes for anticancer drug delivery.

In this article, a novel delivery system for the anticancer drug, arsenic trioxide (ATO), is characterized. The release of ATO from DPPC liposomes with MPPC lysolipid incorporated into the bilayer was measured. Upon heating the liposomes to 37°C, there was a 15-25% release over 24 hours. The ATO release from the DPPC and DPPC:MPPC (5%) systems leveled off after 10 hours at 37°C, whereas the DPPC:MPPC (10%) liposomes continue to release ATO over the 24-hour time span. Upon heating the liposomes rapidly to 42°C, the release rate was substantially increased. The systems containing lysolipids exhibited a very rapid release of a significant amount of arsenic in the first hour. In the first hour, the DPPC:MPPC (5%) liposomes released 40% of the arsenic and the DPPC:MPPC (10%) liposomes released 55% of the arsenic. Arsenic release from pure DPPC liposomes was comparable at 37 and 42°C, indicating that the presence of a lysolipid is necessary for a significant enhancement of the release rate. A coarse-grained molecular dynamics (CGMD) model was used to investigate the enhanced permeability of lysolipid-incorporated liposomes and lipid bilayers. The CG liposomes did not form a gel phase when cooled due to the high curvature; however, permeability was still significantly lower below the liquid-to-gel phase-transition temperature. Simulations of flat DPPC:MPPC bilayers revealed that a peak in the permeability did coincide with the phase transition from the gel to LC state when the lysolipid, MPPC, was present. No pores were observed in the simulations, so it is unlikely this was the permeability-enhancing mechanism.

Factors influencing protective behaviours during haze episodes in Singapore: A population-based study.

INTRODUCTION: Haze is a recurrent problem in Southeast Asia. Exposure to haze is linked to ophthalmic, respiratory and cardiovascular diseases, and mortality. In this study, we investigated the role of demographic factors, knowledge and perceived risk in influencing protective behaviours during the 2013 haze in Singapore. METHODS: We evaluated 696 adults in a cross-sectional study. Participants were sampled via a 2-stage simple random sampling without replacement from a large residential district in Singapore in 2015. The questionnaire measured the participant's knowledge, perceived risk and behaviours during the Southeast Asian haze crisis in 2013. Reliability and validity of the questionnaire were assessed using comparative fit index (≥0.96) and root mean square error of approximation (≤0.05). We performed structural equation modelling to examine the relationship between the hypothesised factors and protective behaviours. RESULTS: More than 95% of the individuals engaged in at least 1 form of protective behaviour. Knowledge was strongly associated with protective behaviours via direct effect (ß=0.45, 95% CI 0.19-0.69, P<0.001) and indirect effect through perceived risk (ß=0.18, 95% CI 0.07-0.31, P=0.002). Perceived risk was associated with protective behaviours (ß=0.28, 95% CI:0.11-0.44, P=0.002). A lower household income and ethnic minority were associated with protective behaviours. A lower education level and smokers were associated with lower knowledge of haze. A higher education and ethnic minority were associated with a lower perceived risk. Wearing of N95 masks was associated with other haze-related protective behaviours (ß=0.24, 95% CI 0.08-0.37, P=0.001). CONCLUSION: Knowledge was associated with protective behaviours, suggesting the importance of public education. Efforts should target those of lower education level and smokers. The wearing of N95 masks correlates with uptake of other protective behaviours.

Free-flow electrophoretic separation and electrical surface properties of subcellular particles from Guinea pig brain.

Continuous free-flow electrophoretic separation has been used to obtain relatively pure preparations of synaptosomes and synaptic vesicles from crude fractions of guinea pig brain homogenates. Measurements of the contents of protein, neuraminic acid, and bound acetylcholine; the activities of succinic dehydrogenase, adenosine triphosphatase, choline acetylase, and 5'-nucleotidase; and the uptake of (14)C-labeled choline arid acetylcholine in the presence and absence of hemicholinium, all confirm the electron microscope evidence that the electrophoretic preparations are at least as pure as those obtained by ultracentrifugal methods. The electrophoretic mobility measurements have been used to calculate zeta potentials and surface charge densities for these particles.

Estrogen formation by the isolated perfused rhesus monkey brain.

Perfusion of two isolated brains from immature male rhesus monkeys with [(3)H]androstenedione resulted in the identification of free and conjugated [(3)H]estrone and free [(3)H]estradiol from the perfusates. In the dissected cerebral tissues, estrogens were recovered only from the hypothalamus and limbic system. The production of estrogens from androstenedione during the 40-minute perfusions in these two experiments totaled 1.58 and 2.83 nanograms.

Prostaglandins and their effects on human placental adenyl cyclase.

Prostaglandins increased adenyl cyclase activity in human term placental homogenates in a dose-dependent manner during 10-min incubation periods. The potency of the prostaglandins examined was demonstrated to be in the ascending order, prostaglandin F(1alpha) < A(2), F(2alpha), B(2) < A(1) < E(2) < E(1). Although no specific trophic or regulating factors for placental function have been described as yet, it is possible that prostaglandins which are synthesized in decidual tissue could play such a physiological role.

Nicotine, cotinine, and anabasine inhibit aromatase in human trophoblast in vitro.

Epidemiologic studies suggest that women who smoke have lower endogenous estrogen than nonsmokers. To explore the possible link between cigarette smoking and decreased endogenous estrogens, we have examined the effects of constituents of tobacco on estrogen production in human choriocarcinoma cells and term placental microsomes. In choriocarcinoma cell cultures, nicotine, cotinine (a major metabolite of nicotine), and anabasine (a minor component of cigarette tobacco) all inhibited androstenedione conversion to estrogen in a dose-dependent fashion. Removal of nicotine, cotinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of aromatase. In the choriocarcinoma cell cultures, a supraphysiologic concentration of androstenedione (73 microM) in the culture medium blocked the inhibition of aromatase caused by nicotine, cotinine, and anabasine. In preparations of term placental microsomes, nicotine, cotinine, and anabasine inhibited the conversion of testosterone to estrogen. Kinetic analysis demonstrated the inhibition to be competitive with respect to the substrate. These findings suggest that some nicotinic alkaloids directly inhibit aromatase. This mechanism may explain, in part, the decreased estrogen observed in women who smoke.

Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle.

The alternative exon 5 of the striated muscle-specific cardiac troponin T (cTNT) gene is included in mRNA from embryonic skeletal and cardiac muscle and excluded in mRNA from the adult. The embryonic splicing pattern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonmuscle cell lines, minigenes express a default exon skipping pattern. Using this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-specific splicing. In this study, we identify four intron elements, one located upstream,and three located downstream of the alternative exon, which act in a positive manner to mediate the embryonic splicing pattern of exon inclusion. Synergistic interactions between at least three of the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites. Mutations in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmuscle cells. Therefore, these elements function as muscle-specific splicing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from the alternative exon is conserved in the rat and chicken cTNT genes. A related sequence is found in a third muscle-specific gene, that encoding skeletal troponin T, downstream from an alternative exon with a developmental pattern of alternative splicing similar to that of rat and chicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number of different genes.

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p.

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

Determinants of ovarian cancer risk. I. Reproductive experiences and family history.

Reproductive experiences and family history were assessed in 215 white females with epithelial ovarian cancer and in 215 control women matched by age, race, and residence. Pregnancy exerted a strong protective effect against ovarian cancer, which increased with the number of live-born children. After adjustment for parity, an effect of age at first live birth and breast-feeding was not apparent. Menstrual events did not differ significantly between cases and controls, although cases were more likely to have had an earlier menopause and less likely to have had a surgical menopause. Women with ovarian cancer had more frequently used menopausal hormones in cyclic fashion compared to controls. Regarding family history, women with ovarian cancer more frequently reported consanguinity in their ancestry and a highly frequency of primary relatives with cancer of the colon, lung, ovary, and prostate gland.

Biochemistry of aromatase: significance to female reproductive physiology.

The formation of estrogens in mammals via aromatase involves the relatively unique capacity to form an aromatic ring de novo in contrast to most other aromatic substances (essential amino acids) which are obtained only in the diet. The reaction is the only example of a cytochrome P450 system which resides in both the mitochondrial and microsomal fractions of the cell. It occurs widely throughout the body in diverse tissues and functions via both de novo synthesis and transformation of prehormones (androstenedione and testosterone). It is found widely in animal species in both the brain and gonads even in phylogenetically primitive species. Placental aromatase appears to be associated with the evolution of viviparity and an extended gestational period in utero. Follicular aromatase which is dependent upon follicle-stimulating hormone stimulation appears to be essential for oogenesis, ovulation, and normal luteal functions while central nervous system aromatase serves to determine sexual behavior and the neurohormonal link to the hypothalamus and pituitary for ovarian cyclicity. While estrogens are the key to pituitary, breast, and endometrial growth and development, this hormone is one of the few examples of an endogenous steroid that has been implicated as a carcinogen or a stimulant for carcinogenesis.

Hormonally induced tumors of the reproductive system of parabiosed male rats.

Parabiosis of intact male rats to castrated males or oophorectomized females for a period of approximately 20 months resulted in three interstitial cell tumors of the testis. When unilateral nephrectomy was added to the parabiotic procedure in ten pairs, eight interstitial cell tumors of the testis and four adenocarcinomas of the prostate occurred in the target male parabionts. These changes were preceded by elevations in luteinizing and follicle-stimulating hormone levels in the serum of the castrates and high levels of testosterone and, to a lesser degree, of androstenedion in the target partners developing the tumors.

Maternal high-fat feeding in pregnancy programs atherosclerotic lesion size in the ApoE*3 Leiden mouse.

Periods of rapid growth seen during the early stages of fetal development, including cell proliferation and differentiation, are greatly influenced by the maternal environment. We demonstrate here that over-nutrition, specifically exposure to a high-fat diet in utero, programed the extent of atherosclerosis in the offspring of ApoE*3 Leiden transgenic mice. Pregnant ApoE*3 Leiden mice were fed either a control chow diet (2.8% fat, n=12) or a high-fat, moderate-cholesterol diet (MHF, 19.4% fat, n=12). Dams were fed the chow diet during the suckling period. At 28 days postnatal age wild type and ApoE*3 Leiden offspring from chow or MHF-fed mothers were fed either a control chow diet (n=37) or a diet rich in cocoa butter (15%) and cholesterol (0.25%), for 14 weeks to induce atherosclerosis (n=36). Offspring from MHF-fed mothers had 1.9-fold larger atherosclerotic lesions (P<0.001). There was no direct effect of prenatal diet on plasma triglycerides or cholesterol; however, transgenic ApoE*3 Leiden offspring displayed raised cholesterol when on an atherogenic diet compared with wild-type controls (P=0.031). Lesion size was correlated with plasma lipid parameters after adjustment for genotype, maternal diet and postnatal diet (R 2=0.563, P<0.001). ApoE*3 Leiden mothers fed a MHF diet developed hypercholesterolemia (plasma cholesterol two-fold higher than in chow-fed mothers, P=0.011). The data strongly suggest that maternal hypercholesterolemia programs later susceptibility to atherosclerosis. This is consistent with previous observations in humans and animal models.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) and serine biosynthetic pathway genes are co-ordinately increased during anabolic agent-induced skeletal muscle growth.

We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20 ppm in feed) or Reporcin (recombinant growth hormone; GH; 10 mg/48 hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p = 0.002) and muscle weights (Vastus Lateralis: p < 0.001; Semitendinosus: p = 0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p < 0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p < 0.05) and 7 (p < 0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p < 0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth.

Factors influencing protective behaviours during haze episodes in Singapore: A population-based study

INTRODUCTION@#Haze is a recurrent problem in Southeast Asia. Exposure to haze is linked to ophthalmic, respiratory and cardiovascular diseases, and mortality. In this study, we investigated the role of demographic factors, knowledge and perceived risk in influencing protective behaviours during the 2013 haze in Singapore.@*METHODS@#We evaluated 696 adults in a cross-sectional study. Participants were sampled via a 2-stage simple random sampling without replacement from a large residential district in Singapore in 2015. The questionnaire measured the participant's knowledge, perceived risk and behaviours during the Southeast Asian haze crisis in 2013. Reliability and validity of the questionnaire were assessed using comparative fit index (≥0.96) and root mean square error of approximation (≤0.05). We performed structural equation modelling to examine the relationship between the hypothesised factors and protective behaviours.@*RESULTS@#More than 95% of the individuals engaged in at least 1 form of protective behaviour. Knowledge was strongly associated with protective behaviours via direct effect (β=0.45, 95% CI 0.19-0.69, @*CONCLUSION@#Knowledge was associated with protective behaviours, suggesting the importance of public education. Efforts should target those of lower education level and smokers. The wearing of N95 masks correlates with uptake of other protective behaviours.

Adenyl cyclase in the human placenta.

PIP: This study demonstrated that the human placenta possesses an adenyl cyclase system responsive to catecholamines and sodium flouride (NaF). 2.5 gm human term placentas were homogenized, centrifuged, washed, resuspended, and used as the enzyme system when placed with various agents. Incubations and the determination of adenosine 3', 5' monophosphate (cyclic AMP) formed were performed. Samples stimulated by .0001 M catecholamines (L-epinephrine or L-norepinephrine) or .01 M NaF had higher levels of cyclic AMP than the controls (p. 005 for catecholamine-treated samples and p. 001 for NaF-treated samples). A concentration of .0001 M L-epinephrine or L-norepinephrine appeared to be a maximum effective dose and .0000001 M a minimum. L=epinephrine was 10 times as effective in the stimulation as L-norepinephrine. With .0001 M, 499 and 439 pmoles/10 minutes per 25 mg of tissue was formed, whereas in the control (no added hormones) 256 pmoles/10 minutes were formed. 3.2% ethanol activated the system by a small amount (p.02). Propranolol alone did not appear to have any effect; however, the effect of .0001 M L-epinephrine was reduced by 95% in the presence of .00001 M propranolol. Propranolol had no effect on NaF-stimulated activity.^ieng

Stimulation of testosterone production in isolated rabbit thecal tissue by LH/FSH, dibutyryl cyclic AMP, PGE2alpha, and PGE2.

The capacities of isolated rabbit theca and granulosa cells to secrete testosterone were studied in vitro. Large Graafian follicles (1-1.5 mm in diameter) were dissected intact from the ovaries of adult estrous rabbits. Granulosa cells from 4 follicles (50,000 cells) and theca tissue (16 pieces per dish, equivalent to 4 follicles) were cultured separately for 6 days either as controls (without exogenous hormones) or with one of the following agents: 1 lU/ml LH/FSH (Pergonal), 10-3M dibutyryl cyclic AMP (Bu2cAMP), 1 mug/ml prostaglandin F2alpha (PGF2alpha), or 1 mug/ml prostaglandin E2 (PGE2). The media were collected every 2 days, and the testosterone (T) was measured by radioimmunoassay. The control cultures of granulosa cells secreted small amounts of T (700 +/- 317 pg/culture: mean +/-SE) during the first 2 days in vitro, and the addition of LH/FSH, Bu2cAMP, PGF2alpha, or PGE2 did not significantly stimulate T production. After 2 days in vitro, very little T (greater than 200 pg/culture) was produced by control and prostaglandin-treated granulosa cells, whereas those incubated with LH/FSH and Bu2cAMP maintained their initial T production rates. Theca control cultures produced 3 +/- 0.4 ng of T (mean +/- SE) during the first 2 days in 13.6-fold by LH/FSH, 3.6-fold by Bu2cAMP, and 3-fold by PGF2alpha and PGE2- T was not detected in theca cultures after 2 days except in those treated with LH/FSH or Bu2cAMP, which produced 1.5 +/- 0.5 and 1.6 +/- 0.3 ng of T, respectively, at 4 days (mean +/- SE). These results suggest that under the present conditions, pieces of rabbit thecal tissue have a greater capacity to produce T de novo than do isolated granulosa cells, and indicate that T production is transiently stimulated by LH/FSH, Bu2cAMP, PGE2alpha, and PGE2.

The effect of LH/FSH, dibutyryl cyclic AMP, and prostaglandins on the production of estrogens by rabbit granulosa cells in vitro.

The purpose of this study was to establish the effects of trophic hormones on the production of estrogens by rabbit granulosa cells. A pure population of these cells was isolated from preovulatory follicles (1-15 mm in diameter) of estrous rabbits, and cultured for 6 days with either one or a combination of the following hormones: androstenedione, Pergonal (LH/FSH), dibutyryl cyclic AMP (Bu2cAMP), prostaglandin F2alpha (PGF2alpha), or prostaglandin E2 (PGE2). The medium was collected every 2 days and progesterone (P), estrone (E1) and estradiol-17 beta (E2beta) were measured by radioimmunoassay. Granulosa cells cultured as controls (i.e., without exogenous trophic hormones) secreted P spontaneously and its secretion was stimulated 100 to 1,000 fold with LH/FSH and Bu2cAMP, but not with PGF2alpha or PGE2. Androstenedione, either alone or with trophic hormones had no apparent effect on the cytology of the granulosa cells or their ability to secrete P. In the absence of exogenous androstenedione, the cultures produced very small amounts of E1 or E2beta (smaller than 100 pg/ml), either spontaneously or in response to LH/FSH, B12cAMP, PGF2alpha, or PGE2. Incubating granulosa cells with exogenous androstenedione (1 mug/ml) resulted in a 30- to 150-fold increase in E2beta production, which was stimulated an additional 3- to 5-fold with LH/FSH and Bu2cAMP, but not with PGF2alpha or PGE2. In most cultures, E2beta production was restricted to the first 2 days in vitro. Bu2cAMP, however, maintained E2beta production at relatively high levels throughout the duration of the experiment, but there was a progressive decrease in its production. The production of E1 was only 5 percent of E2beta, but the pattern of secretion was similar for both estrogens. These results suggest that cyclic AMP could have a role in regulating the synthesis of estrogens by rabbit granulosa cells.

Progesterone, androstenedione, testosterone, estrone, and estradiol synthesis in hamster ovarian follicle cells.

Isolated granulosa cells and theca from proestrous hamsters alone and in recombination, were cultured in order to study steroidogenic capacity of this tissue. Cells from medium size antral follicles (100-300 mum diam.) and large preovulatory follicles (500 plus mum diam.) were used. Steroids were measured by radioimmunoassay. Cultures of cells derived from both sizes of follicles made significant amounts of progesterone for up to 6 days in tissue culture. The preparations from the medium sized antral follicles synthesized little or no estrogen. Of the cells harvested from the preovulatory follicles, the granulosa and theca made moderate amounts of estradiol-17beta while the recombined system made similar to 5 times the estradiol-17beta made by theca or granulosa alone. The results indicate that in the in vitro system used: 1) The hamster follicle cells are similar to other species in that they spontaneously luteinize in culture and secrete large amounts of progesterone, 2) Androgen accumulation is greatest in media from cultured theca of preovulatory follicle, 3) A synergism between theca and granulosa of the large preovulatory follicle exists to effect maximal estrogen synthesis, and 4) Estrogen synthesis is short-lived in vitro in contrast to continued progesterone production.